Description of the Background Art
Immunoassays (and analogous specific binding assays) have revolutionized human diagnostic and veterinary medicine since the introduction of techniques such as the radioimmunoassay (RIA), first reported by Yalow and Berson (Nature 184:1648 (1959)), and the enzyme immunoassay (EIA) which was first reported by Engvall and Perlman (Immunochem. 8:871 (1971)) and Van Weeman and Schuurs (FEBS Lett. 15:232 (1971)).
Immunoassays have enabled exquisitely sensitive measurement of analytes circulating in the blood of a subject, and have allowed the determination of levels of hormones, drugs and other compounds present at very low concentrations (such as picomoles/liter). Such assays, based on antibody-antigen interactions, usually involve complex detection systems. The reagents used are generally antigens labeled with an enzyme or a radioisotope, antibodies or complexes thereof which require either incubation with specific substrates and measurement of a color end-point either visually or by means of a colorimeter, or measurement of radioactive decay with radiation counters to detect the presence of the analyte being tested. These assays also involve several washing steps. Most immunoassays for the detection of analytes in blood are currently of this nature. Although these assays are sensitive, they require lengthy and involved procedures and expensive instrumentation.
An alternative to the RIA and EIA is provided by agglutination immunoassays of the type described by Gupta et al., J. Immunol. Meth. 80:177-187 (1985)), wherein erythrocytes and anti-erythrocyte antibodies are used as the indicator system. Typically, foreign erythrocytes, such as sheep erythrocytes, are used in such immunoassays. Both direct and indirect agglutination assays are known in the art. In the conventional direct assay for an antigen, red cells are coated with antibody, and reacted with the sample. Multifunctional antigens act as bridges between the coated red blood cells, creating an agglutinate. In the conventional indirect assay, red cells are coated with antigen, and contacted with both a soluble antibody and with sample. Sample antigen competitively inhibits the binding of sensitized red cells by the antibody, and hence the agglutination.
Agglutination assays may use other agglutinable particles, For example, latex agglutination assays are described in Castelan et al. (J. Clin. Pathol. 21:638 (1968)) and Singer et al. (Amer. J. Med. [1956 (December)]: 888).
Molinaro, U.S. Pat. No. 4,130,634 describes an agglutination assay employing exogenous red blood cells precoated with antibody. The problem of nonspecific agglutination of erythrocytes by anti-erythrocyte antibodies was noted by Czismas, U.S. Pat. No. 3,639,558, who proposed eliminating all naturally occurring antigenic sites on the red blood cell by coating it with protein. Chang, U.S. Pat. No. 4,433,059 precoated exogenous red blood cells with a covalent, "tail-to-tail" conjugate of an anti-erythrocyte antibody (usually univalent) linked by a heterobifunctional coupling agent to an anti-analyte antibody. See also Smith, W088/05931, Gibbons, U.S. Pat. No. 4,329,011 and Smith, III, U.S. Pat. No. 4,900,685. In the method of the latter patent, the agglutinated erythrocytes are endogenous to the sample. Hillyard, Rylatt, Kemp and Bundesen, U.S. Pat. No. 4,894,347 teach an agglutination immunoassay for whole blood samples featuring the use of endogenous erythrocytes as indicator particles, and of an agglutination reagent in which an erythrocyte binding molecule is conjugated to either an analyte-binding molecule (for a direct assay) or to an analyte analogue (for an indirect assay). They discovered that nonspecific agglutination could be avoided, even when the erythrocyte binding molecule was multivalent, if it recognized an abundant, well distributed membrane constituent such as glycophorin.
In U.S. Pat. No, 4,894,347, we pointed out that in an agglutination assay for antigenic analytes large enough to allow simultaneous binding of two antibody molecules, but which lack repeating epitopes, for agglutination to occur, the antigen must interact with the immunoreagent so that at least some molecules of antigen act as a bridge between proximate erythrocytes. We taught that one solution was to employ a reagent comprising two or more distinct conjugates, i.e., ABM1/EBM+ABM2/EBM, where EBM denotes an erythrocyte binding molecule and ABM1 and ABM2 are analyte binding molecules specific for different, non-overlapping, non-repeating epitopes of the analyte. An agglutination assay of this type is depicted in FIG. 1(b).
This solution, however, has a defect which is particularly apparent when analyte concentrations are low, e.g., for HCG less than 10 nM. That is that a molecule of ABM1/EBM and a molecule of ABM2/EBM can bind simultaneously to a single erythrocyte (FIG. 1(c)). The bound analyte molecule then does not act as a bridge between proximate erythrocytes and therefore does not promote agglutination. Smith, U.S. Pat. No. 4,578,360 and Smith, U.S. Pat. No. 4,401,764 describe conjugates of an erythrocyte binding molecule and a label binding molecule. Chu, U.S. Pat. No. 4,493,793 constructed covalently coupled lectin-antibody or lectin-antigen conjugates. Segal, U.S. Pat. No. 4,676,980 prepared an antibody-antibody immuno therapeutic conjugate for associating a target (e.g., tumor) cell with a cytotoxic effector cell. Freytag, U.S. Pat. No. 4,517,303 describes an immunolytic assay employing a conjugate between an analyte analogue and a cytolysin, e.g., whole mellitin. Li, U.S. Pat. No. 4,661,441 referred to a conjugate of an analyte-binding antibody and an antibody specific for the idiotype of the analyte-binding moiety.
Wardlaw, U.S. Pat. No. 4,695,553 and Guesdon, U.S. Pat. No. 4,668,637 relate to use of "universal" anti-erythrocyte antibodies (and cf. McLaughlin, U.S. Pat. No. 4,683,196), while type-specific antibodies are taught by Lloyd, U.S. Pat. No. 4,678,747, Graham Jr., U.S. Pat. No. 4,358,436, Lu, U.S. Pat. No. 4,550,017, Steplewski, U.S. Pat. No. 4,607,009 and Lennox, W083/03477. Bigtee, Molec. Immunol., 20:1353-1362 (1983) describes the production and testing of anti-glycophorin monoclonal antibodies; Wardlaw suggested use of an anti-glycophorin antibody in clarifying the interface between erythrocytes and leukocytes in centrifuged whole blood. This group of references does not disclose conjugating the erythrocyte binding molecule "tail-to-tail" to another binding molecule.
It is known in the art that a blood sample may be divided into a plurality of aliquots which are then assayed separately for the presence of different analytes. We are not aware of any reference which teaches or suggests dividing a blood sample, reacting the aliquots with different agglutination reagents, and then recombining the sample to obtain in creased sensitivity to a single analyte.
Theofilopoulos (U.S. Pat. No. 4,342,566), Duermeyer (U.S. Pat. No. 4,292,403) and Goldenberg (U.S. Pat. No. 4,331,647) demonstrate the use of antigen binding fragments of antibodies as substitutes for intact antibodies in immunological assays. The construction of heterobifunctional antibodies is taught by Auditore-Hargreaves (U.S. Pat. No. 4,446,233), Paulus (U.S. Pat. No. 4,444,878) and Reading (U.S. Pat. No. 4,474,893). Mochida (U.S. Pat. No. 4,200,436) discloses the use of monovalent antibodies, or antigen-binding fragments thereof, in certain immunoassays. Forrest (U.S. Pat. No. 4,659,678) mentions that monovalent antibodies cannot form dimers or more extensive complexes with the antigen; such monovalent antibody-antigen aggregates were said to be capable of interfering with the binding of the antigen-antibody complex to a solid phase support.
Traditional hemagglutination assays are generally faster, but less sensitive than radioimmunoassays (RIA) or enzyme immunoassays (EIA). The speed and sensitivity of hemagglutination assays can be increased by the use of the autologous red cell agglutination technique described in, e.g., U.S. Pat. No. 4,894,347. The advantage of such a technique is that blood does not have to be separated; a single finger prick is sufficient to provide an assayable sample. For analytes which are large molecules with repeating epitopes, the sensitivity of such an assay is sufficient.
The present inventors recognized a need in the art for improved sensitivity in autologous agglutination assays, in particular for detection of hormones such as human chorionic gonadotrophin (HCG) in the early stages of pregnancy. Such assays with increased sensitivity could also serve as alternatives to inhibition assays in the immunodetection of small molecules. Inhibition assays are inherently less sensitive than direct assays, and the end point is more difficult to define.
No admission is made that any reference cited in this specification is prior art. All references are hereby incorporated by reference.